A thrombus is the result of processes which initiate the coagulation cascade. It is composed of an aggregation of platelets enmeshed in a polymeric network of fibrin. This process is normally initiated as a consequence of tissue injury and has the effect of slowing or preventing blood flow in a vessel. Etiological factors which are not directly related to tissue injury, such as atherosclerotic plaque, inflammation of the blood vessels (phlebitis) and septicemia, may also initiate thrombus formation. In some instances, the inappropriate formation of a thrombus, and subsequent decrease in blood flow, may have pathological consequences, such as stroke, pulmonary embolism and heart disease.
Platelets play a major role in thrombus formation. Current antithrombotic therapy employs agents that modify the platelet/endothelial cell arachidonate-prostaglandin system, such as prostacyclin analogues, cyclooxygenase inhibitors, thromboxane synthesis inhibitors and thromboxane receptor antagonists; and anti-coagulants, such as heparin. These agents inhibit one or both of two discernible phases of platelet aggregation. The primary phase, which is a response to chemical stimuli, such as ADP (adenosine diphosphate), collagen, epinephrine or thrombin, causes initial activation of the platelets. This is followed by a secondary phase, which is initiated by the platelets themselves, and is characterized by thromboxane A.sub.2 (TxA.sub.2) synthesis and the release of additional ADP from platelet storage granules, which further activates platelets.
Platelet aggregation is believed to be mediated primarily through the GPIIb-IIIa platelet receptor complex. Von Willebrand factor, a plasma protein, and fibrinogen are able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and thereby effect aggregation of platelets. Fibronectin, vitronectin and thrombospondin are proteins which have also been demonstrated to bind to GPIIb-IIIa. These proteins, all of which contain an Arg-Gly-Asp peptide sequence, are believed to be members of a superfamily of molecules which mediate cellular adhesion and attachment reactions. Fibronectin, for instance, is found in plasma and as a structural protein in the intracellular matrix. Binding between the structural proteins and GPIIb-IIIa may function to cause platelets to adhere to damaged vessel walls.
Nievelstein et al. (Thromb, and Hemostasis, 58, 2133(1987)) have reported that -RGDS- peptides inhibit thrombin induced aggregation and adhesion of platelets to fibronectin, and may interact through the GPIIb-IIIa complex. U.S. Pat. No. 4,683,291 discloses peptides containing Arg and Lys and an -RGD- sequence which inhibit binding of fibrinogen to platelets and inhibit platelet aggregation. Tetrapeptides which contain the sequence X-Gly-Asp-, wherein X is a guanidine-containing aliphatic carboxylic acid or amino acid residue, are disclosed in EP-A 0 319 506 as inhibitors of platelet aggregation. EP-A 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation. Other peptides and polypeptides which contain an RGD sequence and inhibit fibrinogen binding are disclosed by Plow et al., Blood, 70, 110 (1987), Ginsberg et al., J. Biol. Chem., 260, 3931 (1985), Ruggeri et al., Proc. Natl. Acad. Sci., 83, 5708 (1986) and Haverstick et al., Blood, 66, 946 (1985). Linear and cyclic peptides, the disclosure of which are incorporated herein by reference, are reported in EP-A 0 341 916. There is a need for new molecules which inhibit the binding of GPIIb-IIIa to fibrinogen and inhibit platelet aggregation.
Molecules which mimic a .gamma.-turn in a peptide have been disclosed in attempts to elucidate the biologically active conformation of enkephalin and its analogues. In particular compounds of the formula (I): ##STR1## wherein R and R.sub.1 are H or benzyl and R.sub.2 is H, benzyl or isobutyl, have been disclosed by Huffman et al., Peptides: Chemistry and Biology, Proceedings of the Tenth American Peptide Symposium, Marshall, G. ed., ESCOM, Leiden, 105 (1988). Similar compounds employing a .gamma.-turn mimic have been disclosed by Huffman et al., Synthetic Peptides: Approaches to Biological Problems., UCLA Symposium on Molecular and Cellular Biology, 86, Tam, J. and Kaiser, T. ed., Alan R. Liss, Inc., New York, 257 (1989). These studies concluded that analogues containing these .gamma.-turn mimics did not conform to the biologically active conformation of leucine enkephalin.
It has now been discovered that molecules which mimic the Arg-Gly-Asp sequence and constrain it into a putative .gamma.-turn are useful inhibitors of fibrinogen-GPIIb-IIIa binding and platelet aggregation.
Recent advances for treatment of occluded arteries and deep vein thrombosis employ fibrinolytic agents to lyse thrombi or emboli in order to reestablish or improve blood flow. Fibrinolytic agents, such as anistreplase, tissue plasminogen activator (tPA), urokinase (UK), pro-Urokinase(pUK), and streptokinase (SK), and mutants and derivatives thereof, are proteolytic enzymes which cause fibrin to be hydrolyzed at specific sites and thereby fragment the fibrin network. Lysis of fibrin into smaller peptides has the effect of solubilizing the thrombus or embolus. A recurrent problem with such therapy, however, is the reocclusion of the blood vessel due to formation of a secondary thrombus.
Fibrinolytic therapy is most commonly used for re-establishing flow in a thrombosed blood vessel. However, fibrinolytic therapy does not reverse the factors responsible for the initiation of the thrombus. For this reason, anticoagulants such as heparin are often used to prevent reocclusion. In fact, patients which have a high degree of stenosis in an artery are at extremely high risk of rethrombosis after reperfusion, even in the presence of high doses of heparin. See Gold et al., Circ., 73, 347-52 (1986). In addition, use of SK and tPA has been associated with platelet hyperaggregability. See Ohlstein, et al., Thromb. Res., 4, 575-85 (1987). Treatment with higher doses of tPA can be associated with systemic bleeding and is not recommended for preventing reocclusion. There is, therefore, a need for a method for preventing rethrombosis after fibrinolytic therapy.
EP-A 0 368 232 discloses TxA.sub.2 antagonists for use in a method for inhibiting reocclusion following reperfusion and for lowering the dose of tPA required for fibrinolysis. Yasuda et al. (Clin. Res., 34, 2 (1986)) have demonstrated that reocclusion by fibrin rich platelet thrombi, after thrombolysis with tPA, may be inhibited by a murine monoclonal antibody to GPIIb-IIIa in dogs. This invention discloses a new method for inhibiting reocclusion of a blood vessel by administering compounds which directly inhibit platelet aggregation.